In the present study, we established in vitro and in vivo models to understand mitochondria transfer between astrocytes and glioma cells. We demonstrated that mitochondria are released from normal astrocyte through extracellular vesicles, and are taken up by neighboring glioma cells. Mitochondria transfer not only improves oxidative metabolism in recipient glioma cells, but also supports the detoxification of chemo agent. Mechanistically, mitochondria transfer carries an independent NAD+ metabolic system to the recipient cells, supporting their energy metabolism and NAD synthesis. The improved ATP and NAD synthesis provide essential marcomolecules for PARP-associated base excision DNA repair (BER) pathway. Targeting astrocytic mitochondria releasing pathway improved the cytotoxic effect of chemotherapy, reduced xenograft progression and prolonged overall survival. Our findings suggest that microenvironment-derived mitochondria benefit cancer cells with both metabolic capability and chemo-resistance. Targeting micro-environment-derived mitochondria, combined with other anti-cancer agents, could be a novel avenue in cancer therapies.